A wide variety of nucleic acid molecules with enzymatic or catalytic activity have been discovered in the last 20 years. RNA enzymes (“ribozymes”) occur in nature but can be engineered to specifically recognize and modify a target RNA substrate. In vitro evolution techniques have facilitated the discovery and development of many more catalytic nucleic acids, including deoxyribonucleic acids often referred to as “deoxyribozymes”, “DNA enzymes” or “DNAzymes”. In vitro evolved DNAzymes and/or ribozymes have been discovered which have the capacity to catalyse a broad range of reactions including cleavage of nucleic acids, ligation of nucleic acids, porphyrin metallation, and formation of carbon-carbon bonds, ester bonds or amide bonds.
In particular, DNAzymes and ribozymes have been characterized which specifically cleave distinct nucleic acid sequences after hybridizing via Watson Crick base pairing. DNAzymes are capable of cleaving either RNA or DNA molecules. Ribozymes are also able to cleave both RNA and DNA target sequences. The “10-23” and “8-17” DNAzymes are capable of cleaving nucleic acid substrates at specific RNA phosphodiester bonds to create reaction products which have 2′, 3′-cyclic phosphate and 5′-hydroxyl groups. Examples of deoxyribozymes (DNAzymes), which can ligate 2′, 3′-cyclic phosphate and 5′-hydroxyl products include the “7Z81” and “7Z48” ligases.
More recently, Multi-component Nucleic Acid enzymes (MNAzymes) have been described which have the capacity to self-assemble from two or more oligonucleotide components (also referred to herein as “partzymes”) in the presence of a MNAzyme assembly facilitator (e.g. a target molecule to be detected).
The versatile nature of catalytic nucleic acids has facilitated their use in many different applications. A key element to the successful use of catalytic nucleic acids is their capacity to modify an appropriate substrate. In general, the substrate is substantially complementary to the hybridizing arms of the catalytic nucleic acid and contains a specific sequence or sequence motif at the site of catalytic action. The nature of the interaction between a given catalytic nucleic acid and its substrate is determinative of how efficiently the enzyme engages and/or catalytically modifies its substrate, and is thus a fundamental consideration in designing any system that utilises catalytic nucleic acids.
Catalytic nucleic acids have in vitro diagnostic applications in the detection of nucleic acids, proteins and small molecules. These applications often involve amplification of either the target or the signal to generate sufficient signal for robust detection of the analyte of interest.
Methods that employ catalytic nucleic acids require substrates that are modified with a sufficient rate of catalytic activity to allow effective discrimination over background noise. Different methods may require the use of different reaction temperatures and so there is a necessity for substrates that are efficiently modified (e.g. cleaved) at the required temperatures. Methods such as those utilizing MNAzymes and DNAzymes permit multiplexed analysis of many targets simultaneously in a single reaction, but the ability to multiplex and distinguish between the multiple targets is dependent on the existence of a suitable range of substrates, usually at least one per target. The number of substrates known in the art that are modified (e.g. cleaved) with high efficiency is currently insufficient for mass multiplexing.
The DNAzyme and MNAzyme substrates previously known in the art were derived by screening multiple possible substrates to empirically determine those that were cleaved most efficiently. Often this screening was performed using large numbers of DNAzymes targeted to cleave theoretically possible cleavage sites within full length mRNA. This screening was usually performed under physiological conditions (temperature and ionic strength, composition and pH of buffers). This bias towards finding efficiently cleaved sequences of mRNA at physiological conditions exists because such studies were focused on therapeutic uses of DNAzymes as inhibitors of RNA expression in vivo. Such studies provide a range of laborious protocols for empirical measurement of a large number of putative substrates to find the few that are cleaved efficiently (see for example Cairns et al., 1999 Nat Biotech 17:480-486). These studies resulted in a limited set of design guidelines for the selection of efficiently cleaved substrates, and in many cases the guidelines focused on the design of the DNAzyme rather than the substrate as the DNAzyme can be easily adjusted and the mRNA cannot. One common guideline generated from these studies is that the exact sequence of the R-Y ribonucleotide motif at the cleavage site of the substrate is important with cleavage efficiency being in the following order: GU ≥AU>GC>>>AC.
The efficiency of cleavage of a full length mRNA under in vitro conditions is not an absolute measure of the cleavage efficiency in a cellular environment as the latter includes ribonuclear proteins and other confounding factors that cannot be easily mimicked in vitro.
The design guidelines generated in the past have some use in selection of sites within a long mRNA molecule that may be efficiently cleaved by DNAzymes and MNAzymes under physiological conditions, but have limited ability to predict which substrates will be cleaved with sufficient efficiency for utility in in vitro diagnostic applications. In vitro diagnostic applications may require conditions very different from the physiological conditions generally screened and used to establish the limited substrate design guidelines that exist in the art.
There is a need for a set of guidelines, or sequence motifs, for substrate sequences that predict with greater certainty if a substrate will be efficiently cleaved by a MNAzyme or DNAzyme in conditions suitable for in vitro diagnostic applications. There is also a need for catalytic nucleic acid substrates with properties that facilitate improved catalytic nucleic acid function. These properties may include, for example, an ability to facilitate improved catalytic nucleic acid function over a range of conditions and/or a capacity to extend the number of targets that can be simultaneously detected in a multiplex reaction.